Establishment and maintenance of H19 imprinting in the germline and preimplantation embryo.

The mouse H19 and Igf2 genes are oppositely imprinted and share enhancers that reside 3' to the genes. The imprinted expression of these genes is coordinated by a 2-kb regulatory element, the differentially methylated domain (DMD), positioned between the two genes. The methylation status of this region determines the ability of the insulator factor CTCF to bind to its sites in the DMD. Deletions and mutations of the DMD that affect imprinting in the soma have little effect on the methylation pattern of H19 in the germline, suggesting that additional sequences and factors contribute to the earliest stages of imprinting regulation at this locus. Less is known about these initial steps, which include the marking of the parental alleles, the onset of allele-specific expression patterns and maintenance of the imprints in the preimplantation embryo. Here, we will focus on these early steps, summarizing what is known and what questions remain to be addressed.

Reduced expression of the cyclin-dependent kinase inhibitor gene p57KIP2 in Wilms' tumor.

We have previously shown that the p57KIP2 gene, which encodes a cyclin-dependent kinase inhibitor, undergoes genomic imprinting and lies within a 700-kb domain of imprinted genes on 11p15, including IGF2 and H19. Loss of heterozygosity and loss of imprinting (LOI) of this region are frequently observed in Wilms' tumor (WT) and other embryonal malignancies. Although LOI of p57KIP2 was observed in some WTs (approximately 10%), allele-specific expression was preserved in most tumors examined. Because our initial studies were inconclusive concerning the absolute expression level of p57KIP2 in WT, we developed a sensitive and quantitative RNase protection assay to determine if changes in p57KIP2 expression play a role in WT. Expression of p57KIP2 was found to be virtually absent in 21 of 21 WTs compared to matched normal kidney from the same patients, as well as compared to fetal kidney. We also examined p57KIP2 expression in the normal kidney and tongue of patients with Beckwith-Wiedemann syndrome (BWS), which predisposes to WT and also involves LOI of IGF2 and H19. Although p57KIP2 was undetectable in BWS tongue, similar results were also observed in postnatal non-BWS tongue samples. Most primary skin fibroblast cultures of BWS cell lines exhibited normal imprinting of p57KIP2. However, one BWS patient did show LOI of p57KIP2 in skin fibroblasts. Thus, p57KIP2 is part of a domain of genes on 11p15 that show altered expression and, in some cases, altered imprinting in WT and BWS.

Targeted disruption of Gnas in embryonic stem cells.

Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (G alpha(s)) are present in subjects with Albright hereditary osteodystrophy, a syndrome of characteristic developmental defects and, in some patients, resistance to multiple hormones that stimulate cAMP accumulation (pseudohypoparathyroidism type Ia). As the first step in generating a model of Albright hereditary osteodystrophy, the gene encoding G alpha(s) (Gnas) was disrupted in mouse embryonic stem (ES) cells by homologous recombination. Northern blot analysis and immunoblot analysis demonstrated that steady-state levels of G alpha(s) messenger RNA and G alpha(s) protein in targeted ES cells were approximately 50% of levels in untargeted ES cells. In response to 10 microM forskolin and to various concentrations of isoproterenol (0.1-3.0 microM), cAMP accumulation was reduced in the G alpha(s) knockout ES cell lines, relative to wild-type ES cells and to five of six ES cell lines with randomly integrated targeting vector. These results support the role of G alpha(s) haploinsufficiency in reducing the ability of hormones to generate cAMP in subjects with pseudohypoparathyroidism type Ia. The targeted disruption of Gnas in mouse ES cells establishes an in vitro system for further studies of the role of G alpha(s) and cAMP coupled signal transduction in differentiation and development.

Asymmetric segregation of PIE-1 in C. elegans is mediated by two complementary mechanisms that act through separate PIE-1 protein domains.

The CCCH finger protein PIE-1 is a regulator of germ cell fate that segregates with the germ lineage in early embryos. At each asymmetric division, PIE-1 is inherited preferentially by the germline daughter and is excluded from the somatic daughter. We show that this asymmetry is regulated at the protein level by two complementary mechanisms. The first acts before cell division to enrich PIE-1 in the cytoplasm destined for the germline daughter. The second acts after cell division to eliminate any PIE-1 left in the somatic daughter. The latter mechanism depends on PIE-1's first CCCH finger (ZF1), which targets PIE-1 for degradation in somatic blastomeres. ZF1s in two other germline proteins, POS-1 and MEX-1, are also degraded in somatic blastomeres, suggesting that localized degradation also acts on these proteins to exclude them from somatic lineages.